It is proposed to continue studying the mechanisms of protein degradation in Bacillus subtilis 168 in four areas. (1) Antisera which have now been obtained against both bacillopeptidase F and intracellular serine protease (ISP) will be labeled with ferritin or iodinated. The ferritin-labelled antibodies will be used to try to localize each of the proteolytic enzymes in the cell using electron microscopic techniques. The iodinated antibodies will be used to try to determine the time-course of intracellular appearance of each of these enzymes during sporulation. (2) Studies will be carried out to characterize the recently discovered calcium-activated, subtilisin-dependent peptidase which appears during sporulation. (3) Efforts will be continued on the synthesis of aldehyde analogs of amino-blocked moni, di- and tripeptides. These will be tested both in vitro, against ISP activity and in vivo. (4) Finally, the two-dimensional autoradiograms which are currently being prepared will be analyzed by quantitative densitometry. The autoradiograms are prepared from 2-D polyacrylamide gels bearing 14C- 3H-labelled soluble proteins isolated from B. subtilis during different stages of sporulation. Attempts are being made to measure both 3H and 14C simultaneously.